The present invention relates to a genetic mechanism for mitigating the effects of introgression of a genetically engineered genetic trait of a crop to a weed and of mitigating a weedy potential of the crop and, more particularly, to a genetic mechanism for mitigating the effects of introgression of genetically engineered resistances of crops to weeds.
Crop domestication and weeds: During the prehistoric and historic processes of domestication of crops, farmers selected against a large number of traits that were valuable for wild species, but undesirable in agronomic practice. These differences between wild species and crops were further accentuated by selective breeding, and even more so by genetic engineering, which allowed introducing traits that were non-existent in the gene pool of the species, genus, family, or kingdom of the crop.
Concurrently with domestication, a few wild species evolved to fill the new ecological niches, the disturbed ecosystems known as farmers' fields (Baker, 1974; Holt, 1988; Turner, 1988). Only a few hundred of the tens of thousands of wild species have followed this evolutionary pathway from wild plant to widespread agricultural weed (Holm et al., 1997). Thus, even though some weeds are closely related to crops or are even of the same species as the crops, they vary in a number of traits that distinguish them from wild species, as well as from the crop. These evolutionary processes are not static; indeed they are quite dynamic even on a human generation timescale (Baker, 1991). Changes in agricultural practices (drainage, fertilizer use, tillage and herbicide use) caused some pernicious weeds to return to being wild species, and some wild species to become weeds (Haas and Streibig, 1982). Crops can become “volunteer” weeds in the following crop, or even feral, and re-evolve some weedy traits. Some weeds have even introgressed new traits from conventionally-bred crops (wild barleys in barley have introgressed many new traits; wild sunflowers from sunflowers (Snow et al., 1998). Worse, crops have introgressed weedy traits from related weeds e.g., poor oil quality in canola (Diepenbrock and Leon, 1988), and early bolting in sugar beets from weedy beets (Boudry et al., 1998). These dynamic evolutionary events all occurred before the advent of transgenics.
Crops often possess conventionally-bred traits that would be advantageous to the weeds growing in their midst. Horizontal gene transfer (introgression to totally unrelated species) occurs only rarely to other species within a genus, and even more rarely to species in closely related genera. Thus, even vital traits for weeds such as herbicide resistance have never passed horizontally from non transgenics (Torgersen, 1996), for example from wheat to grass weeds all in the family Poaceae. This lack of horizontal transfer allows the control of these related weeds in the crop. The weeds have had to evolve herbicide resistance from within their own genomes, and not by horizontal gene transfer.
Introgression of genetically engineered traits: The genetic distances between crop and weed were slightly enhanced with the advent of genetic engineering. Traits could be artificially forced horizontally into the crops to enhance cost-effectiveness of agriculture (higher yields, new products, resistances to insects, diseases, and to herbicides). Detractors of both the process of genetic engineering and its products have raised the possibilities that the engineered crops would become uncontrollable weeds, or that the genes would introgress into related weeds, rendering them “weedier”, or into wild species, turning them into weeds (Kloppenburg, 1988; Goldberg et al., 1990; Risler and Mellon, 1993). Hyper-generalizations were raised and terminology such as “superweeds” was coined (Kling, 1996). Calls were issued to prohibit or abandon all transgenic crops because of the possibilities of introgression of such traits into some weeds (Risler and Mellon, 1993). The fact that most crops have no interbreeding relatives in much of the world (Keeler et al., 1996) did not allay the fears of detractors of genetically modified crops. The issues aired in the popular press with extreme statements such as “The greatest danger of genetic engineering of plants may come from sex with weeds”. The debate surrounding introgression of genetically engineered traits has become as sterile as most of the interspecific hybrids generated using highly unnatural lab tricks to save the F1 hybrids (Darmency, 1994).
However, farmers and foresters in most of the world have begun to realize the benefits that accrue from cultivating transgenic crops and forest trees, whether to prevent soil erosion by using post-emergence herbicides or use less expensive/toxic insecticides while contributing to farmer and environmental health and safety. It has been estimated that as much as 60-70% of basic industrial crops (soybean, corn, rapeseed and cotton) used in the US is produced from genetically modified crops (Genetically Engineered Organism-Public Issues Education Project—www.geo-pie.cornell.edu). Additionally, many other traits have been transformed into crops that provide an added value to the crops. Examples of a number of crops, transformed with genes of choice, which have been tested at the field level in the USA, are presented in Table 1.
TABLE 1Primary traits being engineered into major crops field testedin the USA requiring containment and mitigation.CropaPrimary genesaMain introgressional problemsOilseed rapepharmaceuticalsBrassica weeds,herbicide resistanceother oilseed rape,volunteer problemsdisease resistanceinsect resistanceimproved qualitySugar beetherbicide resistanceFeral and wild beets(Carrot/rootdisease resistance(wild carrot)crops)insect resistanceTurf grassherbicide resistancerelated weedsdisease resistanceanti-GMO neighborsinsect resistanceCornherbicide resistancedisease resistanceinsect resistanceagronomic propertiesnutritional qualitypolymerspharmaceuticalsRiceherbicide resistancered (feral) ricedisease resistanceinsect resistancepharmaceuticalsPoplarherbicide resistancenative poplars(Pine)disease resistance(native pines)insect resistancedecreased ligninincrease celluloseaSource: USDA-APHIS website.
Herbicide resistant crops are especially useful for controlling crop-related weeds where there had been no herbicide selectivity. Several crops (e.g., wheat, barley, sorghum, rice, squash, sunflower, sugarbeets, oats, and oilseed rape) can naturally interbreed with closely related weedy relatives under field conditions, in both directions (Gressel, J. 2002: Molecular biology of weed control Taylor and Francis, London; Ellstrand, N. C., Prentice, H. C. & Hancock, J. F. 1999: Gene flow and introgression from domestic plants into their wild relatives. Ann. Rev. Ecol. System. 30: 539-563). There is a concern that transgenes may escape from engineered crops into non-transgenic fields of the same crop, or related weedy or wild species, by hybridization and establish themselves by subsequent backcrossing. This concern has been fueled by the growing number of reports of unintentional, or “accidental” leakage of engineered traits into wild type or weedy crops (Kwon and Sim, Weed Biol and Manag, 2001;1:pg 42; and Hall, et al, Weed Sci 2000; 48:688-94). This could potentially result in large, poorly controlled populations of hybrids and their progeny with enhanced invasiveness or weediness (Ellstrand, N. C., Prentice, H. C. & Hancock, J. F. 1999: Gene flow and introgression from domestic plants into their wild relatives. Ann. Rev. Ecol. System. 30: 539-563; Steward, C. N. Jr., Halfhill, M. D. & Warwick, S. I. 2003. Transgene introgression from genetically modified crops to their wild relatives. Nature Reviews Genetics4:806-817). Many of the engineered genes such as those conferring resistance to herbicides, diseases, and to stresses may grant a fitness advantage to a weedy or wild species. Another increasingly important issue is the concern surrounding transgene flow from crops such as maize bearing transgenes encoding pharmaceuticals to other varieties. Engineered pharmaceuticals, especially vaccines, enzymes and antibodies, can be produced inexpensively in plants, without the need for animal tissue culture cells grown in a medium of expensive serum albumin that is all too easily contaminated with pathogenic mycoplasms, prions and viruses (see, for example, U.S. Pat. No. 6,262,561 to Stewart Jr, et al; U.S. Pat. No. 6,303,341 to Hiatt, et al.; and U.S. Pat. No. 6,395,964 to Arntzen et al, all encorporated herein by reference). Still, there is understandable concern over the introgression of these pharmaceutical transgenes into other varieties of the crop.
Risk analysis and risk mitigation: Tomes have been written on how to assess the risks of introgression—some with continuing generalizations and some discussing how and why this assessment must be undertaken on a case by case basis (Regal, 1994; Keeler et al., 1996; Kareiva et al., 1996; de Kathen, 1998; Williamson, 1993; Timmons et al., 1996; Kjellsson et al., 1998; Sindel, 1997; Gressel and Rotteveel, 2000, Galun and Breman, 1997; Krimsky and Wrubel, 1996). Two general approaches deal with the problems of transgene flow: containment of the transgenes within the transgenic crop, and transgenic mitigation of the effects of the primary transgenic trait should it escape and move to an undesired target. Many containment efforts have depended upon inefficient traditional means such as isolation distances (isolation zones) (see Ritala A., et al, Crop Sci 2002;42:278-85) and barrier crops (see Physical Gene Flow Barriers, page 61; in: Environmental issue report, No. 28, European Environmental Agency Publication No. 28, 2002), less conventional, but still problematic biological means such as apomixis, cleistogamy, male sterility and plastid transformation (see Biological Gene Flow Barriers, pages 60 and 61; in: Environmental issue report, No. 28, European Environmental Agency Publication No. 28, 2002, and Daniell H, Nature Biotechnology, 2002;20:581-86) and the highly complex and uneconomical introduction of lethal traits under control of inducible promoters (see Kuvshinov VV et al, Plant Sci 200; 160:517-522, and U.S. Pat. No. 5,723,765 to Oliver et al). While most containment mechanisms will severely restrict gene flow, some gene flow (leakage) is inevitable and could then spread through the population of undesired species, unless mitigated. Thus, both containment and mitigation strategies are required for efficient and safe use of transgenic crops. Unfortunately, discussions of the hazards and risk assessment have not considered how biotechnologies can be used to mitigate the risk of introgression. No-one, including the governmental panels responsible for authorizing the cultivation of transgenic crops (Anonymous, 1994a, b;, 1997) or those interested in regulatory aspects (Be et al., 1996; Waters, 1996) has seemed to consider the prevention of weeds from using any traits that may introgress from crops, even in the few instances where one can quite surely predict that introgression eventually will occur.
Containing transgene flow—advantages and limitations: Several molecular mechanisms have been suggested to contain transgenes within a genetically modified crop (i.e. to prevent outflow to related species), or to mitigate the effects of transgene flow once it has already occurred (Gressel, 1999, 2002; Daniell, 2002; Steward et al., 2003; Gressel and Al-Almad, 2003). The containment mechanisms include utilization of partial genome incompatibility with crops such as wheat and oilseed rape having multiple genomes derived from different progenitors. When only one of these genomes is compatible for interspecific hybridization with weeds, the risk of introgression could be reduced if the transgene was inserted into the unshared genome where there is presumed to be no homologous introgression between the non-homologous chromosomes. Although it has not yet been reported whether the method of partial genome incompatibility works in wheat, it has been deemed ineffectual for oilseed rape and other similar crops (Tomiuk, J., Hauser, T. P. & Bagger-Jørgensen, R. 2000: A- or C-chromosomes, does it matter for the transfer of transgenes from Brassica napus. Theor. Appl. Genet. 100: 750-754), due to considerable recombination between the A and C genomes.
Another containment strategy is the integration of the transgene into the plastid or mitochondrial genomes (U.S. patent application Ser. No. 20020073443 to Heifetz et al, and Maliga, 2002, and U.S. Pat. Nos. 5,530,191, 5,451,513, 5,932,479, 5,693,507, 6,297,054, 6,376,744, 6,388,168, 6,642,053). The opportunity of gene outflow is limited due to maternal inheritance of these genomes. However, this technology does not prevent the weed from pollinating the crop, and then acting as the recurrent pollen parent. The claim of no paternal inheritance of plastome-encoded traits (Bock, R. 2001: Transgenic plastids in basic research and plant biotechnology. J. Mol. Biol. 312: 425-438; Daniell, H. 2002: Molecular strategies for gene containment in transgenic crops. Nature Biotech. 20: 581-586), has not been substantiated by those authors. Indeed, tobacco (Avni, A. & Edelman, M. 1991: Direct selection for paternal inheritance of chloroplasts in sexual progeny of Nicotiana. Mol. Gen. Genet. 225: 273-277) and other species (Darmency, H. 1994: Genetics of herbicide resistance in weeds and crops, In: Herbicide Resistance in Plants: Biology and Biochemistry, eds. Powles and Holtum, Lewis, Boca-Raton: 263-298) often have between a 10−3-10−4 frequency of pollen transfer of plastid inherited traits in the laboratory. Pollen transmission of plastome traits can only be easily detected using both large samples and selectable genetic markers. A large-scale field experiment utilized a Setaria italica (foxtail or birdseed millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant red leaf base marker) crossed with five different male sterile yellow- or green-leafed herbicide susceptible lines. Chloroplast-inherited resistance was pollen transmitted at a 3×10−4 frequency in >780,000 hybrid offspring. At this transmission frequency, the probability of herbicide resistance from plastomic gene flow is orders of magnitude greater than by spontaneous nuclear genome mutations. Chloroplast transformation is probably unacceptable for preventing transgene outflow, unless stacked with additional mechanisms. For many species, such as pine, the strategy of mitigation via plastome integration is made further improbable due to complete pollen transmission of plastomic traits.
A novel additional combination that considerably lowers the risk of plastome gene outflow within a field (but not gene influx from related strains or species) can come from utilizing male sterility with transplastomic traits [Wang, T., Li, Y., Shi, Y., Reboud, X., Darmency, H. & Gressel, J. 2004: Low frequency transmission of a plastid encoded trait in Setaria italica. Theor. Appl. Genet. 108:315-320]. Introducing plastome-inherited traits into varieties with complete male sterility would vastly reduce the risk of transgene flow, except in the small isolated areas required for line maintenance (see, for example, U.S. Pat. No. 6,372,960 to Michiels et al). Such a double failsafe containment method might be considered sufficient where there are highly stringent requirements for preventing gene outflow to other varieties (e.g. to organically cultivated ones), or where pharmaceutical or industrial traits are engineered into a species. Plastome-encoded transgenes for non-selectable traits (e.g. for pharmaceutical production) could be transformed into the chloroplasts together with a trait such as tentoxin or atrazine resistance as a selectable plastome marker. With such mechanisms to further reduce out-crossing risk, plastome transformation can possibly meet the initial expectations.
Other molecular approaches suggested for crop transgene containment include: seed sterility, utilizing the genetic use restriction technologies (GURT) (U.S. Pat. No. 5,723,765 to Oliver, M. J., et al.), and recoverable block of function (Kuvshinov, V., et al 2001: Molecular control of transgene escape from genetically modified plants. Plant Sci. 160: 517-522). Such proposed technologies control out-crossing and volunteer seed dispersal, but theoretically if the controlling element of the transgene is silenced, expression will occur. Another approach includes the insertion of the transgene under the control of a chemically-induced promoter, so that it will be expressed upon chemical induction (U.S. Pat. No. 6,380,463 to Jepson). However, there remains the distinct possibility of an inducible promoter mutating to become constitutive.
Schernthaner et al (Proc. Natl. Acad. Sci. USA, 2003; 100: 6855-6859) proposed an impractical technology using a “repressible seed-lethal system”. In order to achieve containment, the seed-lethal trait and its repressor must be simultaneously inserted at the same locus on homologous chromosomes in the hybrid the farmer sows to prevent recombination (crossing over), a technology that is not yet workable in plants. The hemizygote transgenic seed lethal parent cannot reproduce by itself, as its seeds are not viable. If the hybrid could be made, half the progeny would not carry the seed-lethal trait (or the trait of interest linked to it) and they would have to be culled, which would not be easy without a marker gene. The results of selfing or cross pollination within the crop and leading to volunteer weeds where 100% containment is needed, would leave only 25% dead and 50% like the hybrid parents and 25% with just the repressor. Thus, the repressor can cross from the volunteers to related weeds as can the trait of choice linked with the lethal, and viable hybrid weeds could form (see U.S. Pat. Pub. No. 200200669425 to Fabijanski et al.). The death of some seed in all future weed generations is inconsequential to weeds that copiously produce seed, as long as the transgenic trait provides some selective advantage.
None of the above containment mechanisms is absolute, but risk can be reduced by stacking containment mechanisms together, compounding the infrequency of gene introgression, possibly stacking a containment that precludes pollination to related species with one that precludes pollination by related species. Still, even at very low frequencies of gene transfer, once such “leakage” occurs, the new bearer of the transgene can disperse throughout the population, if even just a small fitness advantage is conferred to the progeny.
As further detailed hereinunder, a case by case analysis of where intra or interspecific introgressions between genetically engineered crops and weeds are possible shows that there are specific genetic strategies conceivable for mitigation of interspecific introgression.
There is thus a widely recognized need for, and it would be highly advantageous to have, failsafe anti-introgression and introgression-mitigating mechanisms to reduce the possibility of intra and interspecific introgression between genetically engineered crops and weeds.